Total DNA was extracted from muscle by using a DNA isolation kit (Puregene; Gentra, Minneapolis, Minn), and mtDNA was analyzed with Southern blotting as previously described to check for multiple deletions.11 Mutations were detected with single-stranded conformational polymorphism analysis. We used a set of primers that amplified all exons with flanking intronic sequences of POLG, except exon 1, which is not translated (Table 1). Reactions were performed in 25 µL of 10mM Tris hydrochloride (pH 8.9) containing 0.4µM each of the forward and reverse oligonucleotide primers; 1.5 mM of magnesium chloride; 0.2mM each of dATP, dGTP, and dTTP; 0.02mM of dCTP; 1 µCi (37 kBq) of α-32P dCTP; and 1.25 units of Taq DNA polymerase (Roche, Indianapolis, Ind). Polymerase chain reaction (PCR) conditions for exons 4, 5, 6, 7, 8, 9, 10, 22, and 23 were 94°C for 5 minutes, followed by 35 cycles at 94°C for 1 minute, 56°C for 1 minute, 72°C for 1 minute, and a final extension step at 72°C for 7 minutes. For exons 2, 17, and 18, the annealing temperature was 60°C, whereas it was 64°C for exons 11, 12, 13, 14, 15, 16, 19, 20, and 21. Samples were denatured and separated on 6% mutation detection enhancement polyacrylamide gel (Cambrex BioScience Rockland Inc, Rockland, Me) with 5% glycerol, according to the manufacturer's protocol.